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Escalating product recalls as a consequence of Salmonella-contaminated poultry products have resulted in detrimental economic impacts. One long-term alternative to Salmonella prevention, receiving increased attention, is selection to improve genetic resistance. This study evaluated the effects of an oral Salmonella Heidelberg (SH) challenge on bacterial colonization, and the ex vivo effects of SH on phagocytic and bactericidal leukocyte function in turkeys from six pedigree lines (A-F). Data were analyzed using JMP Pro (SAS) and differences were determined using Student’s t-test following ANOVA with significance reported at P ≤ 0.05. Interaction effects of treatment X gender X genetic line were significant on bacterial colonization in the ceca. Cumulatively, females exhibited higher phagocytosis potential than males. The main effect of genetic line was significant bactericidal activity of PBMCs. Microbial profiling of cecal DNA was performed to examine differences in colonization of Salmonella, E. coli, and Enterococcus species among the genetic lines. Results indicated line E having the highest Enterococcus but lowest Salmonella colonization than all other lines, while line A birds displayed the highest Salmonella colonization. These results suggest that gender and genetic line have a marked effect on susceptibility to Salmonella colonization, while genetic line X gender has a more eminent effect on Enterococcus cecal colonization. If able to determine genetic markers associated with these immune responses to Salmonella, genetic selection for increased resistance could be feasible in turkeys.